The present invention relates to the field of high-throughput protein analysis. More specifically, this invention relates to novel reagents for use in the identification and quantitation of proteins using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) in combination with peptide mass fingerprinting or fragment ion-based database searching.
MALDI-MS has become an established tool for the rapid identification of isolated proteins and has been used inter alia to identify proteins involved with human cancers, to elucidate components of multi-protein complexes, as well as for large-scale identification of proteins in organisms with fully sequenced genomes. Prior to MALDI-MS analysis, proteins/peptides are first separated by one-dimensional or two-dimensional polyacrylamide gel electrophoresis (1D or 2D-PAGE) or multidimensional liquid chromatography. Proteins/peptides are then identified by peptide mass mapping or fragment ion based database searching. Analytical procedures involving MALDI-MS are very robust, easy to automate, and most importantly, very fast both in terms of data acquisition and analysis.
However, peptide mass mapping may not routinely yield unambiguous protein identification with high confidence levels, particularly when only a few peptides are encountered. Furthermore, MALDI-MS often yields lower sequence coverage of proteins analyzed than electrospray ionization mass spectrometry (ESI-MS). This lower sequence coverage primarily results from both poor recovery of hydrophobic peptides during sample preparation and inefficient ionization of peptides without arginine residues by MALDI. In addition, MALDI-MS is intrinsically poor as a quantitation tool. Thus, it is very difficult to measure the relative abundance of proteins directly using MALDI-MS data.
There is a need in the art for additional reagents and methods for improving performance of MALDI-MS analysis of proteins/peptides both in terms of confident identification and accurate quantitation.